The goal of this research proposal is to define the molecular mechanisms of cargo identification during the earliest event in the sorting of proteins into the multivesicular body (MVB) pathway. Function of the MVB pathway is critical for a number of cellular phenomena. For example, failure to target signaling growth factor receptors into the MVB pathway, for subsequent degradation in the lysosome ("downregulation"), results in prolonged signaling that can contribute to both tumorigenesis and defects in organismal development. Additionally, defects in downregulation of the epithelial sodium channel result in an inherited form of hypertension (Liddle's syndrome). Major Histocompatability Complex II (MHCII)-mediated immune response relies upon the function of an MVB-like structure, the MHCII-compartment. Aberrant trafficking of lipids through this pathway also contributes to a number of human disease states, including atherosclerosis. Finally, many of the functional components of the MVB pathway are usurped by certain viruses during their life cycle (e.g. HIV-1). We have chosen the model eukaryotic organism Saccharomyces cerevisiae to study the mechanism of cargo identification and sorting into the MVB pathway. We will utilize an ubiquitinindependent MVB cargo to identify cis- and trans-acting factors that coordinate this poorly understood mode of entry into the MVB pathway. Defining the molecular mechanisms that govern MVB cargo identification, a requisite step that dictates cargo entry into the MVB pathway, will yield significant contributions to the understanding of protein and lipid sorting in normal and disease states.